1. Field of the Invention
Nucleic acid hybridization is an excellent tool to identify nucleic acids, and is based upon the principle of complementary base pairing. When single stranded nucleic acids are incubated in solution, complementary base sequences pair to form double-stranded stable hybrid molecules, referred to as DNA complexes. The ability of single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with its complementary nucleotide sequence is often utilized as an analytical tool in recombinant DNA research to identify, isolate and characterize various nucleotide sequences of biological interest.
Detection of gonorrhea is one area in which nucleic acid hybridization holds great potential as a diagnostic tool in clinical medicine. Use of nucleotide probes provides an alternative diagnostic method that can potentially eliminate culturing while simultaneously increase the sensitivity and specificity of an assay for the detection of gonorrhea.
Gonorrhea is one of the most commonly reported bacterial infections. The causative agent, Neisseria gonorrhoeae, is typically identified by culturing on selective agar medium, gram-staining, and cytochrome oxidase and carbohydrate utilization testing. Knapp, Clinical Microbiology Review (1988) 1(4):415-431. U.S. Pat. No. 4,446,230 discloses use of a novel test strain of N. gonorrhoeae in culture detection of N. gonorrhoeae DNA. These tests are usually sufficient to discriminate N. gonorrhoeae from other Neisseria species.
Commercial serological assays, including coagglutination and fluorescent antibody staining, have also been described for the identification of N. gonorrhoeae. Knapp, supra.
This invention relates to novel nucleotide sequences and hybridization procedures for using these sequences. And in particular, this invention relates to nucleotide sequences that are specific for Neisseria gonorrhoeae.
2. Description of the Related Art
Several DNA probe systems have been described for the rapid identification of N. gonorrhoeae. Common to these systems is the use of a nucleotide sequence that is unique to the species N. gonorrhoeae, yet is also capable of recognizing many of its serotypes. A problem in obtaining species-specific probes is overcoming potential cross-hybridization to closely related species. This often requires initial removal of sequences that have homology to the closely related species. There are two subtractive hybridization strategies that have been utilized to enrich for sequences nonhomologous to N. meningitidis in order to isolate the N. gonorrhoeae specific probes. See Welcher et al., Nucleic Acids Research (1986) 14(24):10027-10044 and Donegan et al., Molecular and Cellular Probes (1989) 3:13-26.
Use of a radiolabeled gonococcal cryptic plasmid sequence has been shown to detect 87% of culture-positive N. gonorrhoeae cases. Totten, et al., Journal of Infectious Diseases (1983) 148(3):462-471. These probes are only capable of detecting strains of N. gonorrhoeae which contain plasmid DNA.
Nonradioactive DNA probes have been developed to detect infectious bacteria such as N. gonorrhoeae, Pollice et al., Clinics in Laboratory Medicine (1985) 5(3):463-473. In particular, a biotinylated DNA probe assay for the culture confirmation of N. gonorrhoeae strains is described in Kuritza et al., Diagnostic Microbiology and Infectious Diseases (1989) 12:129-132.
Additionally, nucleic acid probe assays for the noncultural diagnosis of gonorrhea have been developed, such as is described in Kolberg et al., Molecular and Cellular Probes (1989) 3:59-72 which pertains to an assay based upon the DNA sequence of the pilin gene. WO-88/03957 pertains to a nucleic acid probe assay based upon a ribosomal RNA sequence. See also Granato et al., Journal of Clinical Microbiology (1989) 27(4):632-635.
U.S. Pat. No. 4,755,458 discloses the use of a recombinant plasmid consisting of a DNA fragment from N. gonorrhoeae cloned into the vector pBR322, to detect N. gonorrhoeae DNA. The reference generally pertains to a method of detecting polynucleotide sequences.
U.S. Pat. No. 4,900,659 pertains to polynucleotide probe compositions that are capable of hybridizing to N. gonorrhoeae chromosomal DNA. The compositions are obtained from ATCC strains of N. gonorrhoeae.
EP 337896 discloses specific nucleotide sequences useful as probes in detecting Neisseria strains. More generically, EP 227976 pertains to nucleotide probes useful in detecting human infectious diseases such as gonorrhea.